Abstract
Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS/MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain FAs were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification.
Highlights
Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein
We reported that long-chain acyl-CoA synthetase (ACSL) isoform 4 (ACSL4) can use EETs as a substrate [7], but because EET- and HETE-containing phospholipids are present in tissues that express low levels of ACSL4 [8, 9], we wondered whether one or more of the other ACSL isoforms might activate these molecules to form EET- or HETE-CoAs that could be esterified to specific phospholipids
AA is converted to potent eicosanoid signaling molecules by the cyclooxygenase, lipoxygenase, and cytochrome P450 (CYP450) monooxygenase pathways, which produce both EETs and HETEs by CYP epoxygenases and CYP -oxidases, respectively [21]
Summary
Epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification.— Klett, E. The arachidonate (AA) metabolites, epoxyeicosatrienoic acids (EETs) and HETEs, are potent mediators of vasodilation, ion channel activation, anti-inflammatory effects, angiogenesis, mitogenesis, and polypeptide hormone secretion in multiple cell types [1, 2].
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